Supplementary Information Integrated blood barcode chips for rapid multiplexed protein analysis of microliter quantities of blood 1,2,3,5 1,2,3,5 1,4 1,2,3 1,2,3 1,2,3 Rong Fan , Ophir Vermesh , Alok Srivastava , Brian K H Yen , Lidong Qin , Habib Ahmad , Gabriel A Kwong1,2,3, Chao-Chao Liu1,2,3, Juliane Gould1,2,3, Leroy Hood1,4 & James R Heath1,2,3 1NanoSystems Biology Cancer Center, 2Kavli Nanoscience Institute, 3Division of Chemistry and Chemical Engineering, California Institute of Technology, MC 127-72, 1200 E. California Blvd., Pasadena, California, 91125 USA. 4Instititue for Systems Biology, 1441 North 34th St., Seattle, Washington, 98103-8904 USA. 5These authors contributed equally to this work. Correspondence should be addressed to J.R.H. ([email protected]). Supplementary Methods DNA-Encoded Antibody Libraries (DEAL) Technique The critical technique upon which this study is based is the DNA-encoded antibody library (DEAL) method, which has been reported elsewhere by our group1. When DEAL is utilized to measure proteins, it is used as follows (Supplementary Fig. 1). Capture antibodies (CAs) against the protein of interest are chemically labeled with single-stranded DNA (ssDNA) oligomers, yielding ssDNA-CA conjugates. The coupling reaction is accomplished using succinimidyl 4-formylbenzoate (SFB, Solutink) and Succinimidyl 4-hydrazinonicotinate acetone hydrazone in N,N-dimethylformamide (DMF) (SANH, Solulink) as conjugation agents to link amine termini on DNA oligomers to the amine side-groups of proteins1. A size-exchange column is used to purify the product by removing excess unreacted DNA molecules. Separately, the complementary ssDNA oligomers are deposited in a barcode pattern on a poly-L-lysine- coated glass slide using microchannel-guided patterning (details described in Supplementary Fig. 3). At the beginning of a DEAL protein assay, incubation of ssDNA-CA conjugates with the complementary spatially-patterned ssDNA array assembles the CAs onto those specific sites through DNA hybridization. This step transforms the DNA microarray into an antibody 1 Nature Biotechnology: doi: 10.1038/nbt.1507 microarray that is ready for a protein sandwich assay. Biological samples (i.e. plasma isolated from human whole blood) can be applied onto the CA microarray and antigens can be captured. Finally, detection antibodies and/or fluorescent read-out probes are introduced sequentially to complete the immuno-sandwich assay. DNA oligomer sequences are chosen with appropriate melting temperatures to optimize room-temperature hybridization to complementary strands while minimizing cross-hybridization (<5% in fluorescence signal). Supplementary Figure 1 Schematic depiction of multi-parameter detection of proteins in integrated microfluidics using the DNA-Encoded Antibody Library (DEAL) technique. Serum Protein Biomarker Panels and Oligonucleotide Labels The protein panels used in the cancer-patient serum experiment (panel 1) and finger-prick blood test (panel 2), the corresponding DNA codes, and their sequences are summarized in Supplementary Table 1 and 2. These DNA oligomers were synthesized by Integrated DNA Technologies (IDT), and purified by high pressure liquid chromatography (HPLC). The quality was confirmed by mass spectrometry. Supplementary Table 1. List of protein panels and corresponding DNA codes. DNA-code Human Plasma Protein Abbreviation Panel (1) 2 Nature Biotechnology: doi: 10.1038/nbt.1507 A/A’ Interferon-gamma IFN-γ B/B’ Tumor necrosis factor-alpha TNF-α C/C’ Interleukin-2 IL-2 D/D’ Interleukin-1 alpha IL-1α E/E’ Interleukin-1beta IL-1β F/F’ Transforming growth factor beta TGF-β G/G’ Prostate specific antigen (total) PSA H/H’ Interleukin-6 IL-6 I/I’ Interleukin-10 IL-10 J/J’ Interleukin-12 IL-12 K/K’ Granulocyte-macrophage colony stimulating factor GM-CSF L/L’ Monocyte chemoattractant protein -1 MCP-1 M/M’ Blank control/reference Panel (2) AA/AA’ Interleukin-1beta IL-1β BB/BB’ Interleukin-6 IL-6 CC/CC’ Interleukin-10 IL-10 DD/DD’ Tumor necrosis factor-alpha TNF-α EE/EE’ Complement Component 3 C3 FF/FF’ C-reactive protein CRP GG/GG’ Plasminogen Plasminogen HH/HH’ Prostate specific antigen (total) PSA Supplementary Table 2. List of DNA sequences used for spatial encoding of antibodies Sequence Tm (50mM Name Sequence) NaCl) OC A 5'- AAA AAA AAA AAA AAT CCT GGA GCT AAG TCC GTA-3' 57.9 A' 5' NH3- AAA AAA AAA ATA CGG ACT TAG CTC CAG GAT-3' 57.2 B 5'-AAA AAA AAA AAA AGC CTC ATT GAA TCA TGC CTA -3' 57.4 B' 5' NH3AAA AAA AAA ATA GGC ATG ATT CAA TGA GGC -3' 55.9 C 5'- AAA AAA AAA AAA AGC ACT CGT CTA CTA TCG CTA -3' 57.6 C' 5' NH3-AAA AAA AAA ATA GCG ATA GTA GAC GAG TGC -3' 56.2 D 5'-AAA AAA AAA AAA AAT GGT CGA GAT GTC AGA GTA -3' 56.5 D' 5' NH3-AAA AAA AAA ATA CTC TGA CAT CTC GAC CAT -3' 55.7 E 5'-AAA AAA AAA AAA AAT GTG AAG TGG CAG TAT CTA -3' 55.7 E' 5' NH3-AAA AAA AAA ATA GAT ACT GCC ACT TCA CAT -3' 54.7 F 5'-AAA AAA AAA AAA AAT CAG GTA AGG TTC ACG GTA -3' 56.9 F' 5' NH3-AAA AAA AAA ATA CCG TGA ACC TTA CCT GAT -3' 56.1 G 5'-AAA AAA AAA AGA GTA GCC TTC CCG AGC ATT-3' 59.3 G' 5' NH3-AAA AAA AAA AAA TGC TCG GGA AGG CTA CTC-3' 58.6 H 5'-AAA AAA AAA AAT TGA CCA AAC TGC GGT GCG-3' 59.9 3 Nature Biotechnology: doi: 10.1038/nbt.1507 H' 5' NH3-AAA AAA AAA ACG CAC CGC AGT TTG GTC AAT-3' 60.8 I 5'-AAA AAA AAA ATG CCC TAT TGT TGC GTC GGA-3' 60.1 I' 5' NH3-AAA AAA AAA ATC CGA CGC AAC AAT AGG GCA-3' 60.1 J 5'-AAA AAA AAA ATC TTC TAG TTG TCG AGC AGG-3' 56.5 J' 5' NH3-AAA AAA AAA ACC TGC TCG ACA ACT AGA AGA-3' 57.5 K 5'-AAA AAA AAA ATA ATC TAA TTC TGG TCG CGG-3' 55.4 K' 5' NH3-AAA AAA AAA ACC GCG ACC AGA ATT AGA TTA-3' 56.3 L 5'-AAA AAA AAA AGT GAT TAA GTC TGC TTC GGC-3' 57.2 L' 5' NH3-AAA AAA AAA AGC CGA AGC AGA CTT AAT CAC-3' 57.2 M 5'-Cy3-AAA AAA AAA AGT CGA GGA TTC TGA ACC TGT-3' 57.6 M' 5' NH3-AAA AAA AAA AAC AGG TTC AGA ATC CTC GAC-3' 56.9 AA' 5' NH3-AAAAAAAAAAGTCACAGACTAGCCACGAAG-3' 58 BB 5'-AAAAAAAAAAGCGTGTGTGGACTCTCTCTA-3' 58.7 BB' 5' NH3-AAAAAAAAAATAGAGAGAGTCCACACACGC-3' 57.9 CC 5'-AAAAAAAAAATCTTCTAGTTGTCGAGCAGG-3' 56.5 CC' 5' NH3-AAAAAAAAAACCTGCTCGACAACTAGAAGA-3' 57.5 DD 5'-AAAAAAAAAAGATCGTATGGTCCGCTCTCA-3' 58.8 DD' 5' NH3-AAAAAAAAAATGAGAGCGGACCATACGATC-3' 58 EE 5'-AAAAAAAAAAGCACTAACTGGTCTGGGTCA-3' 59.2 EE' 5' NH3-AAAAAAAAAATGACCCAGACCAGTTAGTGC-3' 58.4 FF 5'-AAAAAAAAAATGCCCTATTGTTGCGTCGGA-3' 60.1 FF' 5' NH3-AAAAAAAAAATCCGACGCAACAATAGGGCA-3' 60.1 GG 5'-AAAAAAAAAACTCTGTGAACTGTCATCGGT-3' 57.8 GG' 5' NH3-AAAAAAAAAAACCGATGACAGTTCACAGAG-3' 57 HH 5'-AAAAAAAAAAGAGTAGCCTTCCCGAGCATT-3' 59.3 HH' 5' NH3-AAAAAAAAAAAATGCTCGGGAAGGCTACTC-3' 58.6 * All amine-terminated strands were linked to antibodies to form DNA-antibody conjugates using SFB/SANH coupling chemistry as described by R. Bailey et al.1 Codes AA-HH were used in the experiment which examined fresh whole blood from a healthy volunteer. Codes A-M were used for the molecular analyses of cancer patient serum samples. All matched antibody pairs and standard proteins (recombinants) were received from eBioscience except those described below. The antibody pairs for human C3 and CRP were received from Abcam. Their recombinants were from Sigma. The antibody pair and recombinant protein of human plasminogen were received from Molecular Innovations. The antibody pair for PSA were received from Biodesign. The PSA recombinant was from R&D Systems. The capture and detection antibodies for human hCG were received from Abcam and Chromoprobe, respectively. The antibody pair and the recombinant of human GM-CSF were both received from BD biosciences. All oligonucleotides were synthesized by Integrated DNA Technologies. 4 Nature Biotechnology: doi: 10.1038/nbt.1507 Cross-reactivities of Oligonucleotide Labels A full orthogonality analysis was performed to quantitate the cross-hybridization between the stripes within the DEAL barcode assays. On a barcode array chip, thirteen strands of coding ssDNA (A-M) were patterned. Then, a 13- well PDMS slab was placed onto the barcode chip and in each well a solution containing only one kind of complementary ssDNA from A’-M’ was added. All these complementary strands were labeled with Cy3 and the successful hybridization can be visualized by fluorescence using a 532nm laser excitation. The result (Supplementary Supplementary Figure 2. Cross-hybridization Fig. 2) indicates negligible cross- check for all 13 DNA oligomer pairs that were used hybridization across the entire panel of for encoding the registry of antibody barcode arrays. DNA codes used in our DEAL barcode assay. Patterning of Barcode Arrays Using the microchannel-guided flow-patterning approach (Supplementary Fig. 3), we fabricated DEAL barcode arrays that were ~10-fold denser than conventional microarrays. Microcontact printing can generate high density arrays of biomolecules with spot sizes of a few micrometers (ms)2, 3, but extending stamping to large numbers of biomolecules is awkward because of the difficulty in aligning multiple stamps to produce a single microarray. Direct microfluidics-based pattering of proteins has been reported, but DNA flow-patterning with sufficient loading remains less successful compared to conventional spotting methods4, 5. In the flow patterning process, a polydimethylsiloxane (PDMS) mold containing 13-20 parallel microfluidic channels, with each channel conveying a different biomolecule capture agent, was used.